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Enzo Medico

Publications and source records attributed to Enzo Medico.

9 recordsLinked to original sources
PubMed2007

FLAME, a novel fuzzy clustering method for the analysis of DNA microarray data.

BACKGROUND: Data clustering analysis has been extensively applied to extract information from gene expression profiles obtained with DNA microarrays. To this aim, existing clustering approaches, mainly developed in computer science, have been adapted to microarray data analysis. However, previous studies revealed that microarray datasets have very diverse structures, some of which may not be correctly captured by current clustering methods. We therefore approached the problem from a new starting point, and developed a clustering algorithm designed to capture dataset-specific structures at the beginning of the process. RESULTS: The clustering algorithm is named Fuzzy clustering by Local Approximation of MEmbership (FLAME). Distinctive elements of FLAME are: (i) definition of the neighborhood of each object (gene or sample) and identification of objects with "archetypal" features named Cluster Supporting Objects, around which to construct the clusters; (ii) assignment to each object of a fuzzy membership vector approximated from the memberships of its neighboring objects, by an iterative converging process in which membership spreads from the Cluster Supporting Objects through their neighbors. Comparative analysis with K-means, hierarchical, fuzzy C-means and fuzzy self-organizing maps (SOM) showed that data partitions generated by FLAME are not superimposable to those of other methods and, although different types of datasets are better partitioned by different algorithms, FLAME displays the best overall performance. FLAME is implemented, together with all the above-mentioned algorithms, in a C++ software with graphical interface for Linux and Windows, capable of handling very large datasets, named Gene Expression Data Analysis Studio (GEDAS), freely available under GNU General Public License. CONCLUSION: The FLAME algorithm has intrinsic advantages, such as the ability to capture non-linear relationships and non-globular clusters, the automated definition of the number of clusters, and the identification of cluster outliers, i.e. genes that are not assigned to any cluster. As a result, clusters are more internally homogeneous and more diverse from each other, and provide better partitioning of biological functions. The clustering algorithm can be easily extended to applications different from gene expression analysis.

Algorithms↗
PubMed2006

Neural differentiation of human mesenchymal stem cells: Evidence for expression of neural markers and eag K+ channel types.

OBJECTIVE: Mesenchymal stem cells (MSCs) are multipotent cells that can self-renew, proliferate, and exhibit elevated cellular plasticity. To investigate their possible neural fate, we studied human mesenchymal stem cells (hMSCs) in different cell culture conditions from morphological, immunochemical, gene expression, and physiological points of view. MATERIALS AND METHODS: We tested hMSCs in three previously reported experimental conditions made of alpha-modified minimum essential medium (alpha-MEM)/1 mM beta-mercaptoethanol (betaME), 10 microM alpha-MEM/retinoic acid (RA) or alpha-MEM/2% dimethylsulfoxide (DMSO) + 200 microM beta-hydroxyanisole (BHA), respectively, and in a new experimental condition with neural progenitor maintenance medium (NPMM). RESULTS: hMSCs were isolated from bone marrow and expanded for several passages. In betaME, cells became immunoreactive for neuronal nuclear antigen (NeuN), neuron-specific enolase (NSE), Nestin, and glial fibrillary acidic protein (GFAP). In experimental conditions with RA and DMSO/BHA, hMSCs were NeuN and NSE-positive while in NPMM they were positive for GFAP and NSE. Untreated hMSCs showed a weak mRNA expression for microtubule-associated protein, NSE, and neurofilament protein-medium and GFAP, which strongly increased in NPMM-treated hMSCs. In the electrophysiological study, NPMM-differentiated hMSCs expressed two delayed rectifier K+ currents related to two ether-à-go-go K+ channels (eag1, eag2), which are fundamental for setting the negative resting potentials required for neuronal survival and basal cell activity. The two K+ channels were absent in undifferentiated hMSCs. These data were confirmed by real-time polymerase chain reaction. CONCLUSION: In our new culture condition, hMSCs acquired new morphological characteristics, neural markers, and electrophysiological properties, which are suggestive of neural differentiation. This might lead to clinical use of hMSCs in neural degenerative diseases.

Cell Culture Techniques↗
PubMed2005

The MET oncogene drives a genetic programme linking cancer to haemostasis.

The close relationship between activation of blood coagulation and cancer is an old enigma. In 1865, migrans trombophlebitis ('a condition of the blood that predisposes it to spontaneous coagulation') was described as a forewarning of occult malignancy (Trousseau's sign). This pioneering observation emphasized the existence of haemostasis disorders associated with cancer onset; this phenomenon has since been extensively reported in clinical and epidemiological studies, but has so far resisted a mechanistic explanation. Here we report a mouse model of sporadic tumorigenesis based on genetic manipulation of somatic cells. Targeting the activated, human MET oncogene to adult liver caused slowly progressing hepatocarcinogenesis. This was preceded and accompanied by a syndrome manifesting first with blood hypercoagulation (venous thromboses), and then evolving towards fatal internal haemorrhages. The pathogenesis of this syndrome is driven by the transcriptional response to the oncogene, including prominent upregulation of plasminogen activator inhibitor type 1 (PAI-1) and cyclooxygenase-2 (COX-2) genes. In vivo analysis showed that both proteins support the thrombohaemorrhagic phenotype, thus providing direct genetic evidence for the long-sought-after link between oncogene activation and haemostasis.

Animals↗
PubMed2004

Promoter trapping reveals significant differences in integration site selection between MLV and HIV vectors in primary hematopoietic cells.

Recent reports have indicated that human immunodeficiency virus (HIV) and murine leukemia virus (MLV) vectors preferentially integrate into active genes. Here, we used a novel approach based on genetic trapping to rapidly score several thousand integration sites and found that MLV vectors trapped cellular promoters more efficiently than HIV vectors. Remarkably, 1 in 5 MLV integrations trapped an active promoter in different cell lines and primary hematopoietic cells. Such frequency was even higher in growth-stimulated lymphocytes. We show that the different behavior of MLV and HIV vectors was dependent on a different integration pattern within transcribed genes. Whereas MLV-based traps showed a strong bias for promoter-proximal integration leading to efficient reporter expression, HIV-based traps integrated throughout transcriptional units and were limited for expression by the distance from the promoter and the reading frame of the targeted gene. Our results indicate a strong propensity of MLV to establish transcriptional interactions with cellular promoters, a behavior that may have evolved to enhance proviral expression and may increase the insertional mutagenesis risk. Promoter trapping efficiency provides a convenient readout to assess transcriptional interactions between the vector and its flanking genes at the integration site and to compare integration site selection among different cell types and in different growth conditions.

Cell Line↗
PubMed2004

Autocrine stimulation by osteopontin plays a pivotal role in the expression of the mitogenic and invasive phenotype of RET/PTC-transformed thyroid cells.

Papillary thyroid carcinomas are characterized by rearrangements of the RET receptor tyrosine kinase generating RET/PTC oncogenes. Here we show that osteopontin (OPN), a secreted glycoprotein, is a major RET/PTC-induced transcriptional target in PC Cl 3 thyroid follicular cells. OPN upregulation depended on the integrity of the RET/PTC kinase and tyrosines Y1015 and Y1062, two major RET/PTC autophosphorylation sites. RET/PTC also induced a strong overexpression of CD44, a cell surface signalling receptor for OPN. Upregulation of CD44 was dependent on RET/PTC Y1062, as well. Constitutive OPN overexpression or treatment with exogenous recombinant OPN sharply increased proliferation, Matrigel invasion and spreading in collagen gels of RET/PTC-transformed PC Cl 3 cells. These effects were impaired by the treatment of PC Cl 3-RET/PTC cells with OPN- and CD44-locking antibodies. Thus, RET/PTC signalling triggers an autocrine loop involving OPN and CD44 that sustains proliferation and invasion of transfomed PC Cl 3 thyrocytes.

Carcinoma, Papillary↗
PubMed2004

IGF-II binding to insulin receptor isoform A induces a partially different gene expression profile from insulin binding.

Insulin receptor isoform A (IR-A) is a fetal insulin receptor isoform that is overexpressed in cancer. We investigated whether insulin-like growth factor (IGF)-II may elicit a different gene expression response from insulin in cells expressing only IR-A and lacking IGF-I receptor (R-/IR-A cells). Cells were stimulated with either insulin or IGF-II (at 0.5, 3, and 8 h), and global gene expression was studied by microarray technology. Results were validated by quantitative real-time PCR. We found that 214 transcripts were similarly regulated by insulin and IGF-II, whereas 45 transcripts were differentially regulated. Of these 45 genes, 18 were responsive to only one of the two ligands (12 to insulin and 6 to IGF-II). Twenty-seven transcripts were regulated by both ligands but with a significant difference at at least one time point. IGF-II was a more potent regulator than insulin for these genes. In conclusion, insulin and IGF-II, acting via the same receptor (IR-A), may differentially affect gene expression in cells. These findings provide a molecular basis in clarifying the biological role of IR-A in embryonic/fetal life and in cancer.

Animals↗
PubMed2003

Differential gene expression induced by insulin and insulin-like growth factor-II through the insulin receptor isoform A.

The human insulin receptor (IR) exists in two isoforms (IR-A and IR-B). IR-A is a short isoform, generated by the skipping of exon 11, a small exon encoding for 12 amino acid residues at the carboxyl terminus of the IR alpha-subunit. Recently, we found that IR-A is the predominant isoform in fetal tissues and malignant cells and binds with a high affinity not only insulin but also insulin-like growth factor-II (IGF-II). To investigate whether the activation of IR-A by the two ligands differentially activate post-receptor molecular mechanisms, we studied gene expression in response to IR-A activation by either insulin or IGF-II, using microarray technology. To avoid the interfering effect of the IGF-IR, IGF-II binding to the IR-A was studied in IGF-IR-deficient murine fibroblasts (R- cells) transfected with the human IR-A cDNA (R-/IR-A cells). Gene expression was studied at 0.5, 3, and 8 h. We found that 214 transcripts were similarly regulated by insulin and IGF-II, whereas 45 genes were differentially transcribed. Eighteen of these differentially regulated genes were responsive to only one of the two ligands (12 to insulin and 6 to IGF-II). Twenty-seven transcripts were regulated by both insulin and IGF-II, but a significant difference between the two ligands was present at least in one time point. Interestingly, IGF-II was a more potent and/or persistent regulator than insulin for these genes. Results were validated by measuring the expression of 12 genes by quantitative real-time reverse transcriptase-PCR. In conclusion, we show that insulin and IGF-II, acting via the same receptor, may differentially affect gene expression in cells. These studies provide a molecular basis for understanding some of the biological differences between the two ligands and may help to clarify the biological role of IR-A in embryonic/fetal growth and the selective biological advantage that malignant cells producing IGF-II may acquire via IR-A overexpression.

Animals↗
PubMed2003

Amplification of repeat-containing transcribed sequences (ARTS): a transcriptome fingerprinting strategy to detect functionally relevant microsatellite mutations in cancer.

Cancer is a genetic disease caused by mutations in somatic cells. Those that carry advantageous mutations are favoured by natural selection. In most cancers, genetic instability increases mutation rate and facilitates cancer cell evolution. Microsatellite instability (MSI), due to defects of the DNA mismatch repair system, affects in particular repeat sequences (microsatellites) scattered throughout the genome. As mutations in expressed genes are more likely to be functional, we developed a procedure for the systematic identification of mutant repeat-containing expressed sequences (amplification of repeat-containing transcribed sequences, ARTS). The entire cell mRNA was converted into short double-stranded cDNA fragments linked to an adapter at both ends. Repeat-containing cDNA fragments were PCR amplified using the adapter-specific primer in combination with different arbitrary primers including the repeat. ARTS yielded discrete PCR products with lengths that were directly correlated to the lengths of the endogenous repeats. Comparison between ARTS products obtained from control cells and cancer cells with microsatellite instability (MSI+) revealed mRNAs carrying insertions or deletions at repeats. The subsequent sequencing allowed the identification of a series of frameshift-mutated mRNAs in MSI+ cancer cells, including the already described mutant BAX transcript. These data show that ARTS provides an unbiased genome-wide approach to the discovery of functionally relevant genes that could be affected by MSI in cancer.

Acyltransferases↗